Signaling through costimulatory receptors and ligands is essential for innate and adaptive immunity, and these molecules are validated targets for immune-based therapies to treat a wide range of autoimmune diseases, pathogenic infections and cancer. The genes for costimulatory molecules are often present as tightly linked loci formed by gene duplication events, and as a consequence the members within a given family of costimulatory molecules share common features that are crucial for function. These structural features underlie receptor-ligand recognition, affinity and specificity, oligomeric state, and the formation and localization of ordered signaling complexes, all of which directly impact immune regulation. Defining the shared structural features within the major costimulatory families, as well as the specific differences between family members, provides fundamental mechanistic insight and offers unique opportunities to develop immune-based therapies. In the previous funding period we employed a multidisciplinary approach to study the CD28-B7 families of costimulatory molecules, which included high resolution crystallographic analysis and FRET-based approaches to assay the oligomeric state of wild type and mutant costimulatory molecules on the cell surface. These efforts resulted in the generation of mutant costimulatory molecules with novel ligand binding specificities (PD-1) and unique T cell stimulatory properties (B7-1). Future studies will leverage these findings to determine the roles of the PD-1 and PD-ligand pathways in immunity and pathogenesis. Our recent structural studies on newly described costimulatory molecules, including members of the Signaling Lymphocyte Activation Molecule (SLAM), T cell Immunoglobulin Mucin (TIM) and Tumor Necrosis Factor (TNF) families, have identified unique structural and chemical determinants that define their biological function. Our overall goal remains the utilization of a structure-based approach to gain insight into receptor-ligand recognition and assembly events that represent the first steps in the signal transduction pathways that regulate the immune response. The Specific Aims are: 1) Determination of physical and biochemical features underlying the biological function of the SLAM family of costimulatory molecules;2) Determination of physical and biochemical features underlying the biological function of the TIM family of costimulatory molecules;3) Defining the roles of the PD- 1/PD-Ligand Pathways in Immunity and Pathogenesis;4) Continue the Structural Characterization of the Immune Response. Project Narrative: Signaling through costimulatory receptors and ligands is essential for innate and adaptive immunity, and these molecules are validated targets for immune-based therapies. Defining the shared structural features within the major costimulatory families, as well as the specific differences between family members, provides fundamental mechanistic insight and offers unique opportunities to develop immune-based therapies to treat a wide range of autoimmune diseases, pathogenic infections and cancer.